LOCUS       sequence-432997-        6237 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of 10xHis-SUMO (Smt3) fusion to Sec7 domain 
            (52-252) of Cytohesin-2 (CYTH2/ARNO) for rapid nucleotide exchange 
            and activation of Arf GTPases.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6237)
  AUTHORS   Begley M, Aragon M, Baker RW
  TITLE     A structure-based mechanism for initiation of AP-3 coated vesicle 
            formation.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Dec 24;121(52):e2411974121. doi: 
            10.1073/pnas.2411974121. Epub 2024 Dec 20.
  PUBMED    39705307
REFERENCE   2  (bases 1 to 6237)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2024-12-24"; volume: "121(52):e2411974121. 
            doi"; pages: " 10.1073/pnas.2411974121. Epub 2024 Dec 20"
COMMENT     Sequence Label: MZB150
FEATURES             Location/Qualifiers
     source          1..6237
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             303..332
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1310..1327
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1380..1398)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1394..1441
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1478..1933
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1565..1584)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1775..1796
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2025..2840)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2025..2840)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2748..2767
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2832..2851
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2962..3550
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3451..3470
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3704..3721
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3736..3875
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3801..3820)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3961..3983
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3977..4168)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4977..6059)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6021..6040
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6060..6137)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgg
      301 gccatcacca tcaccatcac catcaccatc acggcagcat gagcgatagc gaagtgaacc
      361 aggaagcgaa accggaagtg aaaccggaag tgaaaccgga aacccatatt aacctgaaag
      421 tgagcgatgg cagcagcgaa atttttttta aaattaaaaa aaccaccccg ctgcgtcgtc
      481 tgatggaagc gtttgcgaaa cgtcagggca aagaaatgga tagcctgcgt tttctgtatg
      541 atggcattcg tattcaggcg gatcagaccc cggaagatct ggatatggaa gataacgata
      601 ttattgaagc gcatcgtgaa cagattggcg gcgccaatga gggcagtaag accttgcaac
      661 ggaaccggaa gatggcaatg ggcaggaaga agttcaacat ggaccccaag aaggggatcc
      721 agttcttggt ggagaatgaa ctgctgcaga acacacccga ggagatcgcc cgcttcctgt
      781 acaagggcga ggggctgaac aagacagcca tcggggacta cctgggggag agggaagaac
      841 tgaacctggc agtgctccac gcttttgtgg atctgcatga gttcaccgac ctcaatctgg
      901 tgcaggccct caggcagttt ctatggagct ttcgcctacc cggagaggcc cagaaaattg
      961 accggatgat ggaggccttc gcccagcgat actgcctgtg caaccctggg gttttccagt
     1021 ccacagacac gtgctatgtg ctgtccttcg ccgtcatcat gctcaacacc agtctccaca
     1081 atcccaatgt ccgggacaag ccgggcctgg agcgctttgt ggccatgaac cggggcatca
     1141 acgagggcgg ggacctgcct gaggagctgc tcaggaacct gtacgacagc atccgaaatg
     1201 agcccttcaa gattccttaa taaggatcca tgggtaccct cgagtgaaga caagcttagg
     1261 tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac ttagtcgagc accaccacca
     1321 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     1381 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     1441 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
     1501 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     1561 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     1621 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     1681 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     1741 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     1801 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     1861 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     1921 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     1981 ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg
     2041 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     2101 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     2161 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     2221 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     2281 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     2341 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     2401 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     2461 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     2521 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     2581 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     2641 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     2701 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     2761 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt
     2821 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt
     2881 attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     2941 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3001 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3061 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3121 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3181 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3241 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3301 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3361 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3421 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3481 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3541 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3601 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3661 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3721 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3781 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     3841 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     3901 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     3961 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     4021 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     4081 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     4141 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     4201 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     4261 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     4321 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     4381 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     4441 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     4501 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     4561 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     4621 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     4681 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     4741 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     4801 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     4861 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     4921 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     4981 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5041 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     5101 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     5161 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     5221 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     5281 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     5341 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     5401 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     5461 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     5521 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     5581 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     5641 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     5701 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     5761 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     5821 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     5881 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     5941 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     6001 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     6061 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     6121 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     6181 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatg
//
